Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Pathology and Laboratory Medicine

Supervisor

Dick, Fred A

Abstract

Therapeutic resistance to androgen receptor inhibitors, such as enzalutamide (EZ), is a major cause of mortality for prostate cancer (PC) patients. Resistance can occur through lineage plasticity, in which PC cells transdifferentiate from an androgen receptor (AR)-dependent to an AR-independent neuroendocrine (NE) cell type. However, the mechanisms that govern therapy resistance are not well defined. Molecular heterogeneity across patients suggests multiple pathways to resistance acquisition exist. This thesis aimed to provide a resource of molecular drivers of EZ resistance and to identify novel targets to treat EZ-resistant PC. Herein, we performed a genome wide CRISPR knockout screen in LNCaP PC cells treated with EZ. This screen identified gene loss events characterized into four functional categories: 1) Essential, 2) Pro-proliferative, 3) EZ-resistant and 4) EZ-sensitive genes. To investigate screen hits in advanced disease, LNCaP cells double knockout (DKO) for RB and p53 were generated, as RB-p53 co-deletion is common in resistant patients. DKO cells are EZ resistant and displayed increased expression of stemness and NE genes, by RNA sequencing. An overlap of screen EZ-sensitivity genes and DKO up-regulated genes from RNA sequencing identified the transcription factor HOXA9. Depletion or chemical inhibition HOXA9 sensitized cells to EZ, whereas its overexpression drove EZ resistance. Our screen identified the transcriptional repressor TBX18 as an EZ-resistant gene. TBX18 mutation promoted EZ resistance and is associated with poor prognosis in PC patients. The transcription factor SOX4 scored as an essential gene in our screen. SOX4 deficiency hindered cell growth, whereas SOX4 overexpression in DKO cells drove EZ resistance. Finally, we investigated the applicability of HOXA9 as a biomarker of EZ resistance. Immunohistochemical staining for HOXA9 and its downstream target genes SOX4 and FLT3 were optimized and validated on prostate tumour specimens. Positive and negative expression in tumours sections were observed for each marker. Altogether, our CRISPR screen provides a resource of molecular mediators of EZ resistance in PC. We validated several novel drivers that can potentially serve as biomarkers and molecular treatment targets. Currently, no targeted therapeutics are available for resistant patients, so there is a need to identify novel treatment strategies to combat this disease.

Summary for Lay Audience

In advanced prostate cancer (PC), the development of resistance to treatments, such as inhibitors like enzalutamide (EZ) that target the androgen receptor (AR), is a major cause of death for patients. However, the genes and molecular pathways involved in causing therapeutic resistance are not well defined. Previous studies show that multiple genes and pathways can lead to treatment resistance, suggesting that different patients may benefit from different treatment strategies. To provide a resource of potential genes involved in the development of resistance to EZ and to identify new potential treatment targets, we performed a screen using CRISPR-Cas9 to determine whether mutations within almost any gene in the human genome can affect basic cell growth or response to EZ treatment. In addition, we used a model of advanced disease, EZ-resistant PC cells that have gene knockouts for both RB1 and TP53 (DKO), as mutations in both genes commonly occur in EZ-resistant patients. By comparing genes that scored in our screen and genes that were highly expressed in DKO cells, we implicated HOXA9 and SOX4 in treatment resistance. We showed that reduced expression or inhibition of HOXA9 with a preclinical drug can sensitize PC cells to EZ, whereas overexpressing this protein makes cells EZ-resistant. Although SOX4 mutation primarily hindered basic cell growth, we also showed that overexpressing this protein in DKO cells caused these cells to become even more EZ-resistant. Increased expression of HOXA9 and SOX4 are both associated with advanced disease and poor prognosis in PC patients. We also validated that gene mutation of TBX18, another highly scoring gene in our screen, drove EZ-resistance. Finally, we developed antibody tissue staining protocols as a proof of principle to show that HOXA9 is expressed in some prostate cancer patient samples and has the potential to be used as a diagnostic tool to identify patients at potentially higher risk of developing resistance. Overall, this thesis provides a resource and validation of several genes we newly implicated in causing resistance to EZ in PC cells, which may serve as novel markers and targets for the treatment of EZ-resistant patients.

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Available for download on Thursday, June 18, 2026

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